ASSIGNMENT OF THE ALIPHATIC H-1 AND C-13 RESONANCES OF THE BACILLUS-SUBTILIS GLUCOSE PERMEASE-IIA DOMAIN USING DOUBLE-RESONANCE AND TRIPLE-RESONANCE HETERONUCLEAR 3-DIMENSIONAL NMR-SPECTROSCOPY

Nearly complete assignment of the aliphatic H-1 and C-13 resonances of the IIA(glc) domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the H-1(alpha) and C-13(alpha) chemical shifts of one residue with the amide N-15 chemical shift of the following residue, was used to obtain sequence-specific assignments of the C-13(alpha) resonances. The H-1(alpha) and amide N-15 chemical shifts had been sequentially assigned previously using principally 3D H-1-N-15 NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-69071. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their H-1(alpha) and C-13(alpha) chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional H-1-H-1 correlation experiments that rely on H-1-H-1 3J couplings, which, for proteins the size of IIA(glc) (17.4 kDa), may be significantly smaller than the H-1 line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIA(glc) domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D H-1-N-15 NOESY-HMQC and H-1-C-13 NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].